Identification of 4′-Thiouridine as an Orally Available Antiviral Agent Targeting Both RdRp and NiRAN Functions of SARS-CoV‑2 Nsp12

Through screening of a nucleos(t)ide-focused chemical
library,
we identified 4′-thiouridine (1) as a potent antiviral
agent against SARS-CoV-2 in Vero cells, with an EC50 value
of 1.71 μM and a CC50 value exceeding 100 μM.
Its triphosphate metabolite, compound 8, inhibited the
RNA-dependent RNA polymerase activity of the SARS-CoV-2 Nsp12-Nsp7-Nsp82 complex, terminating nascent RNA synthesis through misincorporation.
Additionally, compound 8 suppressed the function of the
NiRAN domain of Nsp12, effectively blocking both RNAylation and NMPylation
of Nsp9. Pharmacokinetic analysis in mice showed excellent oral bioavailability
of compound 1. Oral administration at 100 mg/kg/day,
twice daily for 5 days, protected mice from lethal SARS-CoV-2 infection,
resulting in 40% survival and near-complete recovery of body weight
by day 14 postinfection. Compound 1 also exhibited broad-spectrum
activity against various coronaviruses and other RNA viruses. These
findings highlight that compound 1 is a promising orally
available antiviral candidate.